Facile Induction of Immune Tolerance by an Interleukin-2-TGF-ß Surrogate Agonist

CD4? regulatory T (Treg) cells are essential for establishing and maintaining immune tolerance. Peripherally induced Tregs (pTregs), which differentiate from mature conventional CD4? T cells in the periphery, complement Tregs generated in the thymus during T cell development by broadening Treg reactivity in response to changing self and the environment. Although both TGF-ß and Interleukin-2 (IL-2) synergistically promote functional pTreg development in vitro, their combined roles in inducing pTreg generation in vivo have not been thoroughly characterized and therefore are yet to be exploited for tolerizing immunotherapy. The principal limitation to in vivo use of IL-2 and TGF-ß is their pleiotropy and inability to ensure co-delivery of SMAD and STAT signaling to the same cell. To overcome these challenges, we designed an IL-2-TGF-ß surrogate agonist by creating a fusion protein between IL-2 and a low-affinity TGF-ß mimic agonist derived from a Helminth parasite. This IL-2-TGF-ß surrogate agonist was easily expressed and purified and enabled simultaneous cis-activation of both IL-2 and TGF-ß signaling specifically in IL-2 receptor-expressing T cells. This IL-2-TGF-ß surrogate agonist robustly induced antigen-specific, functional, and stable pTregs in vivo within peripheral lymphoid organs in OVA- or MOG35-55-immunized mice. The induced pTregs phenotypically resemble effector and colonic Ror?t? pTregs, with the potential to efficiently migrate into the gut and suppress intestinal inflammation. Treatment with this agonist effectively quelled immune activation in mouse models of allergen-induced allergic inflammation and self-antigen-driven autoimmune neuroinflammation, portending a strategy for the targeted induction of antigen-specific pTregs in vivo to induce immune tolerance in inflammatory and autoimmune diseases. Overall design: One million sorted Thy1.1? naïve OT-II cells were adoptively transferred into Thy1.2? Foxp3-GFP recipient mice via retro-orbital intravenous injection. This was followed by six intraperitoneal injections of 100 µg OVA protein combined with 50 pmol of the indicated proteins, administered every other day. On day 11, donor OT-II cells and host Foxp3-GFP? CD4? T cells were sorted from the mLNs of the respective treatment groups and submitted to MedGenome, Inc. for library preparation and RNA sequencing.